This competitive renewal application will test three novel hypotheses about the interactions between human polymorphonuclear leukocytes (PMN) and ingested Staphylococcus aureus (SA) and their implications for the fate of human PMN, SA infection and inflammation. A significant fraction of ingested SA survives in PMN and prompts PMN to initiate cell fate programs that result in atypical apoptosis. These apoptotic, SA-laden PMN bind to macrophages (Mf), skew Mf cytokine profiles, and block efferocytosis, by incompletely defined mechanisms. SA-laden PMN not internalized by Mf undergo necrotic cell death and lysis, thereby releasing viable SA that are more virulent after passage through PMN. Within the framework of our novel, integrated view of microbicidal events in PMN, we will determine the transcriptional responses of SA that mediate their survival in PMN, define signaling pathways initiated by PMN in response to viable intracellular SA that trigger the atypical and eventual necrotic cell death seen in SA-infected PMN. Lastly, we will determine the mechanistic basis for and consequences of the failure of Mf to clear PMN-SA and the skewed cytokine profile of Mf that ingest PMN in which viable SA persist. The latter studies will provide important insight into how SA undermine cellular events that normally culminate in resolution of inflammation and return to tissue homeostasis. Each of the inter-related but independent aims is supported by strong preliminary data. Aim 1 Determine the mechanisms and consequences of survival of SA in human PMN phagosomes We will determine how SA upregulation of msrA1/B, YjiE, and other transcriptional responses to HOCl and other PMN- generated toxins promote SA survival (1.A.i-iii) and the functional consequences to PMN and Mf (1.B.). Aim 2 Determine mechanisms for functional changes in PMN and Mf during SA infection We will identify the signalling pathways and determine the molecular basis for atypical apoptosis of PMN-SA (2.A.) -- focusing on Mcl-1 (2.A.i), IL-1b (2.A.ii), and HIF-1a (2.A.iii) -- the decreased efferocytosis of PMN-SA by Mf and its impact on promoting inflammation (2.B), and the eventual necrotic cell death of PMN-SA (2.C). Our studies will identify mechanisms responsible for the atypical apoptosis and necrotic death of PMN, thereby providing novel insights into fundamental aspects of phagocyte control of the inflammatory response. From the microbial perspective, we will identify the functional consequences of transcriptional adaptations of surviving SA that were induced by PMN exposure, thus providing insight into SA vulnerability that could be exploited as novel targets for therapeutic attack.